Clinical experience and perinatal outcome of blastocyst transfer after coculture of human embryos with human endometrial epithelial cells: a 5-year follow-up study

OBJECTIVE: To evaluate the reproductive and neonatal outcome of blastocyst transfer after coculture with human endometrial epithelial cells in IVF and oocyte donation. DESIGN: Retrospective study.Private assisted reproductive center. PATIENTS(S): Two hundred sixty women undergoing IVF and 469 oocyte recipients. INTERVENTION(S): IVF or intracytoplasmic sperm injection (ICSI) and transfer of at least one blastocyst after coculture with human endometrial epithelial cells. MAIN OUTCOME MEASURE(S): Blastocyst formation rate, implantation and pregnancy rates, neonatal outcome, and congenital birth defects. RESULT(S): Among patients who had transfer with their own oocytes, 1193 of 2349 cocultured embryos developed up to the blastocyst stage (50.8%), and pregnancy and implantation rates of 33.9% and 19.2%, respectively, were achieved. In the oocyte donation program, 1819 blastocysts were obtained from 3127 embryos (58.2%), with subsequent pregnancy and implantation rates of 57.0% and 31.0%, respectively. The blastocyst rate remained stable throughout the 5 years of the study, but the pregnancy and implantation rates increased dramatically. Of 139 deliveries, 57 (41.0%) were multiple pregnancies and 1 (0.7%) was a multifetal birth (four live born infants). Out of 200 children born, 59% were male, and congenital birth defects were observed in 2.5%. CONCLUSION(S): Coculture of human embryos with endometrial epithelial cells yields a blastocyst formation rate of 50.8% to 58.2% and encouraging implantation and pregnancy rates. This technique reduces the mean number of embryos transferred in each patient. The number of embryos implanted is more relevant to neonatal outcome than is the coculture system and blastocyst transfer used. The risk of congenital birth defects associated with this program is similar to that recorded in early ET in IVF or ICSI.     

Cycle dependency of intrauterine vascular endothelial growth factor levels is correlated with decidualization and corpus luteum function

OBJECTIVE: To determine intrauterine levels of vascular endothelial growth factor (VEGF)-A during the menstrual cycle in the human female and to investigate the impact of decidualization and corpus luteum function. DESIGN: Prospective clinical study. SETTING: Tertiary university center. PATIENT(S): Fifty-four women with infertility problems. INTERVENTION(S): Intrauterine concentrations of VEGF-A were determined at various time points during the secretory phase using a novel intrauterine microdialysis device. Concomitantly, intrauterine insulin-like growth factor binding protein (IGFBP)-1 levels served as a paracrine parameter for decidualization. Serum progesterone (P) and E(2) levels were determined as markers for corpus luteum function.Intrauterine VEGF levels. RESULT(S): The VEGF levels in utero were clearly cycle dependent with increasing levels during the late secretory and premenstrual phases. There was a significant correlation with the decidualization marker IGFBP-1. In contrast, intrauterine VEGF levels showed a significant negative correlation with serum E(2) and P. CONCLUSION(S): Intrauterine VEGF levels are regulated in a cycle-dependent way. Increasing levels in the late secretory phase are clearly correlated with decidualization. In contrast, decreasing serum levels of steroids produced by the regressing corpus luteum are less likely to be responsible for increasing VEGF levels in the premenstrual phase.     

Effects of initiation day of clomiphene citrate on the endometrium of women with regular menstrual cycles

OBJECTIVE: To investigate the effects of initiation time of clomiphene citrate (CC) on the endometrium of women with regular menstrual cycles. DESIGN: Randomized, double-blind, cross-over study. SETTING: Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. PATIENT(S): Thirty-three healthy female volunteers with regular menstrual cycles. INTERVENTION(S): The volunteers were randomized to receive either 100 mg of CC on days 1-5 and placebo on days 5-9 (study group) or placebo on days 1-5 and CC on days 5-9 (control group). After a wash-out period of 1 month of CC treatment, the medication was switched in each group. Ultrasonography was performed daily after day 10 of the cycle to detect ovulation. Ultrasonography for endometrial appearance and thickness, endometrial sampling, and blood samples obtained for determination of E(2) and P levels were performed 7 days after ovulation in both groups. MAIN OUTCOME MEASURE(S): Morphometric analysis, histologic dating, and ultrasonographic appearance and thickness of the endometrium. RESULT(S): Morphometric parameters, histologic dating, and ultrasonographic appearance and thickness of the endometrium were similar in both groups. CONCLUSION(S): Starting CC on either day 1 or day 5 of the menstrual cycle did not have any differential effects on the endometrium of women with regular menstrual cycles, particularly regarding the morphometric analysis, histologic dating, or ultrasonographic appearance.     

Hydrosalpinx fluid diminishes endometrial cell HOXA10 expression

OBJECTIVE: To determine the effect of hydrosalpinx fluid on the expression of HOXA10, an essential regulator of endometrial receptivity.DESIGN: In vitro study.SETTING: Academic medical center.PATIENT(S): Patients with unilateral or bilateral hydrosalpinx.INTERVENTION(S): Hydrosalpinx fluid was aspirated from 10 patients at laparoscopy. The fluid was serially diluted in minimum essential medium. Ishikawa cells (an endometrial adenocarcinoma cell line, representative of endometrial epithelium) were incubated with this fluid at concentrations of 10% and 50% for 48 hours. Cells were also incubated in undiluted minimum essential medium (MEM) and in 10% serum as controls. After incubation, the cells were lysed in Trizol, and total RNA was extracted and analyzed by Northern blot using a 32P-labeled HOXA10 riboprobe. A 32P-labeled G3PDH probe was used as a control for loading.MAIN OUTCOME MEASURE(S): HOXA10 mRNA expression.RESULT(S): HOXA10 mRNA expression in endometrial cells decreased with increasing concentrations of hydrosalpinx fluid. Densitometric analysis of the northern blot revealed that HOXA10 mRNA expression was different from control at both concentrations (P<.007).CONCLUSION(S): HOXA10 is necessary for implantation in the murine model. HOXA10 expression is diminished by hydrosalpinx fluid. This effect on HOXA10 is a potential molecular mechanism by which implantation rates are diminished in women with hydrosalpinges.     

The alpha(2)beta(1) and alpha(3)beta(1) integrins do not mediate attachment of endometrial cells to peritoneal mesothelium

OBJECTIVE: To evaluate the possible role of mesothelial alpha(2)beta(1) and alpha(3)beta(1) integrins in the attachment of endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs). DESIGN: In vitro study. SETTING: University medical center. PATIENT(S): Women of reproductive age (n = 26). MAIN OUTCOME MEASURE(S): Mesothelial cells were grown on collagen IV. Endometrial stromal cells and EECs were plated on mesothelial cells for 1 hour. Before plating, mesothelial cells or endometrial cells were incubated with antibodies to alpha2, alpha3, and beta1 integrin subunits. The effect of these antibodies on ESC and EEC binding to collagen IV and collagen I was also examined. The expression of collagen I, collagen IV, fibronectin, and laminin by cultured ESCs and EECs was examined. RESULT(S): The anti-integrin antibodies had no effect on endometrial binding to mesothelium. The beta1 integrin antibody decreased binding of ESCs and EECs to the collagen matrices. In culture, ESCs and EECs expressed collagen I, collagen IV, fibronectin, and laminin to varying degrees. CONCLUSION(S): The initial adhesion of ESCs and EECs to mesothelium is not mediated by beta1 integrins. In contrast, ESC and EEC attachment to collagen IV and collagen I, which are present in the submesothelial extracellular matrix, is mediated by beta1 integrins.     

Prospective study on gynaecological effects of two antioestrogens tamoxifen and toremifene in postmenopausal women

To assess and compare the gynaecological consequences of the use of 2 antioestrogens we examined 167 postmenopausal breast cancer patients before and during the use of either tamoxifen (20 mg/day, n = 84) or toremifene (40 mg/day, n = 83) as an adjuvant treatment of stage II-III breast cancer. Detailed interview concerning menopausal symptoms, pelvic examination including transvaginal sonography (TVS) and collection of endometrial sample were performed at baseline and at 6, 12, 24 and 36 months of treatment. In a subgroup of 30 women (15 using tamoxifen and 15 toremifene) pulsatility index (PI) in an uterine artery was measured before and at 6 and 12 months of treatment. The mean (+/-SD) follow-up time was 2.3 +/- 0.8 years. 35% of the patients complained of vasomotor symptoms before the start of the trial. This rate increased to 60.0% during the first year of the trial, being similar among patients using tamoxifen (57.1%) and toremifene (62.7%). Vaginal dryness, which was present in 6.0% at baseline, increased during the use of tamoxifen (26.2%) and toremifene (24.1%). Endometrial thickness increased from baseline (3.9 +/- 2.7 mm) to 6.8 +/- 4.2 mm at 6 months (P< 0.001), and no difference emerged between the 2 regimens in this regard. Before the start of the antioestrogen regimen, the endometrium was atrophic in 71 (75.5%) and proliferative in 19 of 94 (20.2%) samples; 4 patients had benign endometrial polyps. During the use of antioestrogen altogether 339 endometrial samples were taken (159 in tamoxifen group, 180 in toremifene group). The endometrium was proliferative more often in the tamoxifen group (47.8%) than in the toremifene group (32.2%) (P< 0.0001). 20 patients had a total of 24 polyps (17 in tamoxifen and 9 in toremifene group, P< 0.05) during the use of antioestrogens. One patient in the toremifene group developed endometrial adenocarcinoma at 12 months, and one patient had breast cancer metastasis on the endometrium. Tamoxifen failed to affect the PI in the uterine artery, but toremifene reduced it by 15.0% (P< 0.05) by 12 months. In conclusion, tamoxifen and toremifene cause similarly vasomotor and vaginal symptoms. Neither regimen led to the development of premalignant endometrial changes. Our data suggest that so close endometrial surveillance as used in our study may not be mandatory during the first 3 years of use of antioestrogen treatment.     

Is endometrial pre-treatment of value in improving the outcome of transcervical resection of the endometrium?

The aim of this study was to determine whether or not the use of medical pre-treatment of the endometrium improves the outcome of transcervical resection of the endometrium with regards to long-term operative outcome, histological findings and patient satisfaction. A prospective randomized trial comparing three endometrial pre-treatment agents (danazol, medroxyprogesterone acetate or nafarelin) with no pre-treatment was conducted. The main outcome measures were: (i) thickness of the endometrium and myometrium resected; (ii) histological stage of the endometrium at the time of operation; (iii) the presence or absence of menses and (iv) patient satisfaction 1 year post-operatively. Of the three pre-treatments studied, danazol produced a lower median endometrial thickness than the control, showed the greatest ability to induce atrophy of the endometrial glands and stroma (not statistically significant) and produced the highest rate of amenorrhoea (not different to the control). Danazol and nafarelin produced significantly lower median endometrial thickness than no pre-treatment. There were, however, no significant differences in the rates of amenorrhoea in any of the pre-treatment groups compared with that in the control group. No improvement in clinical outcome or patient satisfaction is conferred by the use of medical pre-treatments if transcervical resection of the endometrium is performed in the proliferative phase of the menstrual cycle.     

The measurement of endometrial perfusion in norplant users: a pilot study

The use of progestogens without oestrogen is commonly associated with irregular menstrual bleeding. Oestrogens and progestogens are both thought to influence endometrial perfusion; changes in endometrial perfusion may contribute to vascular fragility and breakdown. In this study, endometrial perfusion was measured using laser-Doppler fluxmetry in women in the secretory phase of the menstrual cycle before and 4-6 weeks after insertion of the low-dose long-acting levonorgestrel contraceptive implant system, Norplant. Endometrial perfusion was also measured in women exposed to Norplant for up to 19 months. There was no significant difference between endometrial perfusion in control cycles (27.2 flux units +/- 5.5, SEM) and at 4-6 weeks after Norplant insertion (16.3 flux units +/- 5.0), a time when irregular bleeding and spotting are common. Endometrial perfusion was no different from controls after longer periods of Norplant exposure (35.7 flux units +/- 7.2). No direct relationships between endometrial perfusion and plasma concentrations of ovarian steroid hormones were demonstrated. Short-term endometrial vasomotion was largely abolished during Norplant exposure.     

Spatially regulated differentiation of endometrial vascular smooth muscle cells

Angiogenesis within the human endometrium involves the development of arterioles and elaboration of a capillary network. It was postulated that maturation of these arterioles involves a spatially regulated process of vascular smooth muscle cell (VSMC) differentiation. The endometrial vascular tree was therefore examined immunohistochemically for evidence of longitudinal and radial gradients of VSMC phenotype. Twenty-three hysterectomy specimens and 15 first trimester decidual tissues were studied. Five cytoskeletal markers (alpha and gamma-smooth muscle (sm) actin, sm myosin, desmin, vimentin), three endothelial markers (CD31, CD34, factor VIII related antigen) and two steroid receptors (oestrogen and progesterone) were detected immunohistochemically. alpha-sm actin was present throughout the wall of basal arterial segments and extended longitudinally towards the endometrial surface. Sm myosin expression was more restricted longitudinally and radially within in the vascular tree. The expression of gamma-sm actin was even more restricted than myosin. In first trimester decidua, however, gamma-sm actin was widely distributed within the wall of spiral arteries that were not invaded by trophoblast. Oestrogen and progesterone receptors were present in peri-vascular stromal cells but absent from vascular smooth muscle and endothelium. Endometrial VSMC differentiation involves a progressive increase in cytoskeletal complexity and occurs in a spatially regulated fashion.